Post release modification of viral envelopes

ABSTRACT

The present invention relates to enveloped virus particles providing a modified composition of their envelope, methods for exogenously modifying the envelope composition of an enveloped viral particle making use of compounds consisting of a hydrophilic target domain and a lipophilic membrane anchor domain, wherein the lipophilic membrane anchor domain becomes anchored into the lipid double layer of the envelope and wherein the hydrophilic target domain becomes exposed to the surrounding incubation fluid. The invention further relates to methods and means to use said modified viral vectors and to pharmaceutical compositions containing such envelope modified viral vectors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and is a U. S. national stage ofInternational application number PCT/EP2009/054016 filed Apr. 3, 2009,which claims the benefit of European application number EP 08154748.1filed Apr. 17, 2008, each of which is hereby incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention relates to enveloped virus particles providing analtered composition of viral envelopes, methods and means forexogenously altering the composition of viral envelopes by anchoring ofsubstances into viral membranes, pharmaceutical compositions and the useof such altered enveloped virus particles for medical, biotechnological,research or diagnostic purposes.

BACKGROUND OF THE INVENTION

Cholesterol-rich microdomains like rafts and caveolae are specializedregions of the plasma membrane and play an important role for severalcellular processes e.g. signal transduction, and for the life cycle ofcertain viruses (e.g., the entry and exit steps). These domains areenriched in cholesterol, sphingomyelin, ganglioside GM1 and caveolinproteins. The cholesterol molecules are intercalated between the lipidacyl chains and cause a decrease in the fluidity of these membraneregions leading to their resistance against treatment with non-ionicdetergents like Triton X-100. Therefore, these regions are also referredto as detergent resistant microdomains (DRMs). The specific lipidcomposition of DRMs leads to the selective incorporation andconcentration of specific cellular proteins. Such proteins often exhibitGlycosylphosphatidylinositol (GPI) anchoring and/or fatty acylation.

It is known that viral proteins of certain viruses e.g. the envelopeprotein (Env) of the ecotropic murine leukemia virus (E-MLV) or of thehuman immunodeficiency virus type 1 (HIV-1) associate with DRMs aftertransport to the plasma membrane (Beer (2005), Brügger (2007)).Similarly, Gag proteins of HIV-1 prefer DRMs as cellular destinationsafter synthesis in the cytoplasm. Mutations of HIV-1 Env or E-MLV Envpalmitoylation sites or the HIV-1 Gag myristylation site impair theassociation of these proteins with DRMs. Moreover, knock out of Envpalmitylation sites led to a decreased viral titer due to a reduced Envincorporation into the viral particles.

Therefore, DRMs enriched in viral capsid and envelope proteins areconsidered to be platforms for assembly and budding of viruses frominfected cells.

The virus envelope on the other hand consists of envelope proteins (Env)embedded in a membrane consisting of a lipid double layer, whereas theviral membrane composition in some cases resembles the lipid compositionof DRMs and it is believed that it differs from the average distributionof lipids in the plasma membrane of the host cell. For HIV-1 it wasshown that the viral nef protein modulates the lipid composition of DRMsin infected T-lymphocytes (Brügger (2007)). It is also known that theviral envelope contains cellular proteins picked up from the virusproducing cell during the budding process. This has been used toincorporate specific membrane proteins e.g. complement inhibitors likeCD59 as described, in WO 00/17374 into envelops of viral particlesmaking use of the natural transport pathway of the host cell.

Recently such approaches have also been used to increase theimmunogenicity of simian immunodeficiency virus (SIV) based virus-likeparticles (VLPs) by addition of CD40 ligand and granulocyte-macrophagecolony-stimulating factor (GM-CSF) or to modify murine leukaemia virus(MLV) based VLPs with cytokines such as IL-4 to induce differentiationof monocytes, or IL-2 to induce proliferation of peripheral bloodmononuclear cells (PBMCs).

Modification of retroviral vectors so far depends on the geneticengineering of virus producer cell lines e.g. by transfection orinfection. Such an approach is very inconvenient and time consuming,because for each component to be inserted into viral envelops therespective host cell producing the virus has to be genetically modifiedand even then there are no guarantees that the modifications willmanifest in the correct way on the virus. Such an approach is alsolimited to proteins and polypeptides which have to be synthesized by thehost cell and transported to the plasma membrane.

A typical cellular transport pathway makes use of aglycosylphosphatidylinositol (GPI) anchor to direct proteins andpolypeptides to the outer leaflet of cell membranes. GPI anchors areattached to protein precursors at the endoplasmatic reticulum (ER)membrane by a transamidase enzyme complex and delivered to the outerleaflet of the plasma membrane. GPI-linked proteins serve differentfunctions i.e. in the regulation of complement activity (e.g. CD59;CD55) or as hydrolytic enzymes (e.g. alkaline phosphatase or renaldipeptidase). GPI anchors consist of a hydrophilic oligosaccharide and alipophilic fatty acid part.

It was already demonstrated that the lipophilic part of GPI anchors maymediate the exogenous insertion of proteins into lipid membranes ofcells (Medof (1996); Legler (2005); McHugh (1995); Premkumar (2001)) andartificial lipid membranes (Ronzon (2004); Morandat (2002)) which aregenerally characterized by a low protein content in relation to thefatty acid content. The process of exogenous insertion of substanceslike proteins into cell membranes was termed “painting” (Medof (1996)).It has further been demonstrated that GPI anchors can be added topreviously non-GPI-linked proteins (e.g. green fluorescent protein (GFP)by genetically addition of a GPI signaling sequence (GSS) to theirC-terminal end and that these proteins retain their biological functions(McHugh (1995); Premkumar (2001); Legler (2005)).

In contrast to artificial or cellular membranes the amount of viralenvelope proteins in virus envelopes is extremely high so that thelipids of the virus envelope are typically shadowed making viruses e.g.highly resistant to detergents and environmental influences. Typicallipid to protein mol fractions in cell membranes expressed as molepercentage (mol %) are 80:20 or higher whereas in typical viralenvelopes the ratio is 30:70 or less (M. K. Jain, R. C. Wagner:Introduction to Biological Membranes, New York (Wiley) 1980, ISBN0471034711) The accessibility of the virus membrane lipid layer isdrastically reduced which makes it unlikely that large voluminousproteins or other voluminous compounds can be inserted into the viralenvelope after release of the virus from its host cell. While trying togenerate more efficient therapeutic viral vectors for gene therapy andvaccination, the inventors developed methods for an easier and morerapid anchoring of substances into membranes of enveloped viruses,especially in order to provide a quick and flexible alternative toengineering of genetically modified virus producing cell lines.Furthermore, the inventors were also able to anchor compounds other thansole proteins or polypeptides into membranes of enveloped viruses.

It was absolutely unexpected and surprising for the inventors thatcompounds, especially proteins linked to membrane anchor domains likeglycosylphosphatidylinositol (GPI) anchors can successfully be insertedinto lipid double layers of viral envelopes when added exogenously toisolated viral particles resulting in viral particles with alteredsurface characteristics. It was further surprising that envelopedviruses which are generally sensitive to lysis remain stable andinfective after treatment with GPI proteins that can form largermicelle-like structures which are expected to disrupt the virusenvelope. The method presented herein is useful for generation of virusparticles with well designed chemical and biological characteristicsdepending on the target domains/moieties inserted.

DETAILED DESCRIPTION OF THE INVENTION

The terms “virus”, “virion” and “viral particle” are usedinterchangeably. A virus is a submicroscopic infectious agent that isunable to grow or reproduce outside a host cell. Each virus consists ofgenetic material, DNA or RNA, within a protective protein coat called acapsid. The capsid shape varies from simple helical and icoshedral(polyhedral or near-spherical) forms, to more complex structures withtails or an envelope. In the context of the present invention onlyenveloped viruses are considered. Viruses infect cellular life forms andare grouped into animal, plant and bacterial types, according to thetype of host infected. For the correct functioning of the invention, thegenetic information of the viral particle is not relevant. Therefore,the present invention comprises wild-type viruses, attenuated viruses,empty virus particles as well as genetically modified viral vectorswhich can either be replication competent or incompetent.

The viral vector may contain additional modifications to its naturalviral envelope e.g. by genetic modification of the viral genome toproduce e.g. chimeric envelope proteins or by making use of packagingcell lines for pseudotyping, as used for infection retargeting and/orcapping proteins, as used to prevent virus infection or to limit it tocertain cell types able to uncap the protein. Virus infectioncharacteristics could also be modulated by painting virions withproteins that modulate envelope distribution (e.g. patching). Suchmodifications of viral envelopes are known in the art and are commonlyused to alter the viral tropism.

The viral painting process according to the present invention simplifiesthe procedure for the generation of such viruses and facilitates the useof various combinations of these approaches to modify infectionspecificities of viruses and virus derived vector systems.

For example patent application WO2005118802 describes retroviralvectors, particularly lentiviral vectors, pseudotyped with a geneticallymodified Sindbis virus envelope and targeted to specific cell types viaa ZZ domain of protein A linked to the envelope.

Recent publications have demonstrated that for retroviral vectors,target binding and fusion function of envelope proteins may be separatedby introducing binding independent fusogenes of e.g. Sindbis virus (YangL (2006, 2008), Yang H (2008), Ziegler (2008)).

Binding specifies can be provided in a second step i.e. by paintingaccording to the present invention thus providing a high degree offlexibility (a basic vector carrying the fusogene may be equipped with abroad range of different binding molecules)

Another possibility to post-modify viral envelope proteins of virionsalready released from its host cell is to alter the viral envelopeproteins chemically or biochemically.

For example in WO93/09221, influenza virus tropism was modified byinhibition of the viral hemagglutinin polypeptide which normallymediates the binding of the virus to the cellular receptor by means of amonoclonal antibody and by coupling the virus with an antibody capableof interacting with the transferrin receptor expressed onto targetedcells.

Roux (1989) reported the infection of human cells with a mouse ecotropicrecombinant retrovirus using two biotinylated antibodies directed to theretroviral envelope gp70 and to a cellular antigen of the human majorhistocompatibility complex (MHC), respectively.

Such modifications to the viral envelope might additionally beintroduced into the viral particle either prior to, subsequent to orsimultaneously with the painting procedure of the present invention.

An “enveloped virus” is a virus which exhibits a viral envelope. A viralenvelope typically has a protein to lipid mole fraction expressed inmole percentage (mol %) between 50 and 90, preferably 65 to 85 and mostpreferably 70 to 80. The term “enveloped virus” according to the presentinvention comprise following taxonomic families, which can be divided incorresponding subfamilies, genus and species, whereas only not limitingrepresentative examples of species are shown. An “enveloped virus” canbe selected from any group of family, subfamily, genus or species.

The classification is in accordance with the “VIIIth Report of theInternational Committee on Taxonomy of Viruses, 2005” (C. M. Fauquet, M.A. Mayo, J. Maniloff, U. Desselberger, and L. A. Ball (eds), VirusTaxonomy, Academic Press, 1162 pp. (2005)) which is herein incorporatedby reference. Preferably the “enveloped virus” is a poxvirus, aherpesvirus or a retrovirus, more preferably a gamma retrovirus or alentivirus, most preferably mouse leukemia virus (MLV) and/or FelineHerpesvirus-1 (FHV-1).

Family Subfamily Genus Species Arenaviridae Arenavirus Lymphocyticchorio- meningitis virus Lassavirus Bunyaviridae OrthobunyavirusBunyamwera virus Hantavirus Hantaan virus Nairovirus Dugbe virusKrim-Kongo virus Phlebovirus Rift Valley fever virus CoronaviridaeCoronavirus Infectious bronchitis virus Torovirus Equine torovirusFiloviridae Marburgvirus Lake Victoria Marburg virus Ebolavirus Zaireebolavirus Flaviviridae Flavivirus Yellow fever virus Pestivirus Bovineviral diarrhea virus 1 Hepacivirus Hepatitis C virus HepadnaviridaeOrthohepadnavirus Hepatitis-B-Virus Avihepdnavirus DuckHepatitis-B-Virus Herpesviridae Ictalurivirus Ictalurid herpesvirus 1 Alphaherpesvirinae Simplexvirus Human herpesvirus 1 Feline herpesvirus1 Varicellovirus Human herpesvirus 3 Mardivirus Gallid herpesvirus 2Iltovirus Gallid herpesvirus 1  Betaherpesvirinae Cytomegalovirus Humanherpesvirus 5 Muromegalovirus Murid herpesvirus 1 Roseolovirus Humanherpesvirus 6  Gammaherpesvirinae Lymphocryptovirus Human herpesvirus 4Rhadinovirus Saimiriine herpesvirus 2 Orthomyxoviridae Influenzavirus AInfluenza A virus Influenzavirus C Influenza C virus ThogotovirusThogoto virus Dhori virus Influenzavirus B Influenza B virus IsavirusInfectious salmon anemia virus Paramyxoviridae  ParamyxovirinaeRespirovirus Sendai virus Morbillivirus Measles virus Rubulavirus Mumpsvirus Henipavirus Hendra virus Avulavirus Newcastle disease virus Pneumovirinae Pneumovirus Human respiratory syncytial virusMetapneumovirus Avian metapneumo- virus Poxviridae  ChordopoxvirinaeOrthopoxvirus Vaccinia virus Parapoxvirus Orf virus Avipoxvirus Fowlpoxvirus Capripoxvirus Sheeppox virus Leporipoxvirus Myxoma virusSuipoxvirus Swinepox virus Molluscipoxvirus Molluscum contagiosum virusYatapoxvirus Yaba monkey tumor virus  EntomopoxvirinaeAlphaentomopoxvirus Melolontha melolontha entomopoxvirusBetaentomopoxvirus Amsacta moorei entomopoxvirus ‘L’ GammaentomopoxvirusChironomus luridus entomopoxvirus Retroviridae  OrthoretrovirinaeBetaretrovirus Mouse mammary tumour virus Jaagsiekte sheep retro- virusLangur virus Mason-Pfizer monkey virus Squirrel monkey retro- virusGammaretrovirus Murine leukemia virus Feline leukemia virus Gibbon apeleukemia virus Guinea pig type-C onco- virus Porcine type-C onco- virusFinkel-Biskis-Jinkins murine sarcoma virus Gardner-Arnstein felinesarcoma virus Hardy-Zuckerman feline sarcoma virus Harvey murine sarcomavirus Kirsten murine sarcoma virus Moloney murine sarcoma virusSnyder-Theilen feline sarcoma virus Woolly monkey sarcoma virus Viperretrovirus Chick syncytial virus Reticuloendotheliosis virus Trager duckspleen necrosis virus Alpharetrovirus Avian leukosis virus Rous sarcomavirus (RSV) Avian myeloblastosis virus Avian carcinoma Mill Hill virusAvian myelocytomatosis virus 29 Avian sarcoma virus CT10 Fujinamisarcoma virus Deltaretrovirus Bovine leukemia virus Human T-cell lympho-tropic virus type I Human T-cell lympho- tropic virus type II SimianT-cell lympho- tropic virus type I Simian T-cell lympho- tropic virustype II Lentivirus Human immunodefi- ciency virus 1 Human immunodefi-ciency virus 2 Simian immunodefi- ciency virus (SIV) Bovine immunodefi-ciency virus (BIV) Jembrana Disease Virus Equine infectious anemia virus(EIAV) Feline immunodefi- ciencyvirus (FIV) Maedi visna virus (MVV)Caprine arthritis enceph- alitis virus Epsilonretrovirus Walleye dermalsarcoma virus Walleye epidermal hyper- plasia virus 1 Walleye epidermalhyper- plasia virus 2  Spumaretrovirinae Spumavirus Simian foamy virusFeline foamy virus Equine foamy virus Bovine foamy virus RhabdoviridaeVesiculovirus Vesicular stomatitis Indiana virus Lyssavirus Rabies virusEphemerovirus Bovine ephemeral fever virus Novirhabdovirus Infectioushematopoietic necrosis virus Togaviridae Alphavirus Sindbis virusRubivirus Rubella virus

“Exogenous” in the context of the present invention means that thecompounds to be inserted into the virus envelope are added to isolatedenveloped viruses in an appropriate suspension medium like body fluid,buffered saline or cell culture medium e.g. DMEM. The presence of acellular host is not necessary for the process/technology. The method istermed “viral painting”.

The terms “altered” or “modified” viral particle, virion or virus in thecontext of the present invention are used interchangeably. All termsrefer to enveloped viruses wherein the composition of the viral envelopeis modified by anchoring of compounds into the lipid double layer of thevirus envelope after release (post release) of the viral particle fromits respective host cell, preferably after isolation and concentrationof intact viral particles from its natural environment or a technicalproduction process. The modification of the composition of the virusenvelope can be used to modify the chemical and/or biologicalcharacteristics of a virus particle. For example the stability ofviruses against sheering forces or the resistance to detergents can beadjusted. Also important is the possibility to modify the infectivity,affinity or the host spectrum of the virions, allowing for a specificdesign of therapeutic viral vectors e.g. with pre-designed tissuespecificity or vaccines that preferentially infect professional antigenpresenting cells. Anchoring of protein marker and/or compounds havingmoieties with specific chemical or physical properties can be used toenhance the detectability and/or sensitivity of diagnostic methods.Linkage of molecules with immunological functions, most notablycytokines, by the viral painting process can provide a range of novelproperties to the virion e.g. stimulation of the immune system. Paintedenveloped viral vectors can be turned into specific adjuvants forvaccination approaches through an increased immunogenicity. Theflexibility and the speed of the modular viral painting will beespecially useful for vaccination approaches against viruses thatfrequently change or rearrange their antigens (e.g. influenza) or aredifficult to target (e.g. HIV-1). Cytokines are a category of signalingmolecules that, like hormones and neurotransmitters, are usedextensively in cellular communication. They are proteins, peptides orglycoproteins.

The term “cytokine” encompasses a large and diverse family ofpolypeptide regulators that are produced widely throughout the body bycells of diverse embryological origin.

Cytokines may be divided into six groups: interleukins,colony-stimulating factors, interferons, tumor necrosis factor, growthfactors, and chemokines.

Interleukins are proteins that are produced mainly by lymphocytes ormacrophages and act on other leukocytes. At least 18 types with varyingorigin and function exist.

Colony-stimulating factors are produced by lymphoid and nonlymphoidcells. These factors provide a mechanism whereby cells that are distantfrom bone marrow can call for different types of hemopoietic progeny.There are also growth-promoting actions of locally producedcolony-stimulating factors within the bone marrow to stimulateprogenitors to differentiate into macrophages, granulocytes, or coloniescontaining both cell types.

Interferons classically interfere with the virus replication mechanismsin cells. Interferon-□ (produced by leukocytes) and interferon-□(produced by fibroblasts) activate cytotoxicity in natural killer cells.Interferon-□ also activates natural killer cells, and is a potentactivator of macrophages as well.

Tumor necrosis factor-□ (TNF-□) is produced by a variety of cell types,but activated macrophages represent the dominant source. TNF-□ activatesnatural killer cell cytotoxicity, enhances generation of cytotoxicT-lymphocytes, and activates natural killer cells to produceinterferon-□ TNF-□ also acts on vascular endothelium to promoteinflammation and thrombosis. TNF-□ may also induce apoptosis in cellssuch as trophoblasts. TNF-□ is a product of Th1 T-cells; in addition toproviding help in proinflammatory cell-mediated immune responses, thesecells produce delayed-type hypersensitivity reactions where macrophagesare locally recruited and activated to kill intracellular pathogens,such as certain bacteria. TNF-□ as interferon-type activity and anarrower spectrum of action than TNF-□

Transforming growth factors (TGFs) have the ability to promoteunrestrained proliferation of cells which otherwise has a benignbehavior phenotype. These factors have therefore been implicated indevelopment of cancer. There are two groups of transforming growthfactors. TGF-□ is a 5-kilodalton peptide produced by a variety of cellsand collaborates with TGF-□, a 25-kD peptide, in promoting unrestrainedtumorlike growth. TGF-□ has potent pleiotropic effects on a wide varietyof tissues and is a potent fibrogenic and immunosuppressive agent.

Chemokines are chemoattractant cytokines of small (7-14 kD)heparin-binding proteins that are subdivided into four families: CXC,CC, C, and CX3C. Chemokines are produced by macrophages stimulated bybacterial endotoxins, and control the nature and magnitude of cellinfiltration in inflammation.

Preferred cytokines according to the present invention are interleukinsand colony-stimulating factors, especially interleukin-1 (IL-2),interleukin-4 (IL-4), granulocyte-macrophage colony stimulating factor(GM-CSF) and/or Interleukin-12 (IL-12), preferably of human origin.

For painting purposes according to the present invention fusion proteinsconsisting of cytokines and membrane anchor domains are used. Thecytokines are preferably fused at their C-terminus to a membrane anchordomain which preferably is GPI. The C-terminal stop codon of thecytokine is hereby replaced by a short linker sequence e.g.poly-glycine.

A “compound” according to the present invention is a chemical substancewhich is capable of inserting or integrating itself into lipid doublelayers, especially of viral envelopes. Such compounds consist of atleast two distinct parts: (a) a membrane anchor domain or moiety and,(b) a hydrophilic target domain or moiety to be exposed to the outsideof the viral particle. Such compounds may contain further parts,providing for additional physical or chemical properties or to allow forlinkage to other materials such as nanoparticles or beads. Compounds maycontain also protein-tags such as the histidine tag and/or flag-tag,that is important for purification of said compounds or for purificationand/or concentration of viral particles modified with said compounds.

“Membrane anchor domains or moieties” are amphiphilic molecules whichare able to integrate into lipid double layers with their lipophilicpart, whereas the hydrophilic part is exposed to the surrounding waterymedium. The hydrophobic group is typically a large hydrocarbon moiety,such as a long chain of the form R═CH3(CH2)n, with n>4 or a cyclichydrocarbon moiety. The hydrophilic group can either be charged orpolar, uncharged groups. Charged groups are e.g. carboxylates: RCO2−;sulfates: RSO4−; sulfonates: RSO3−. phosphates (the typical chargedfunctionality in phospholipids), carbohydrates or amines RNH3+. Polar,uncharged groups are alcohols with large R groups, such as diacylglycerol (DAG), and oligoethyleneglycols with long alkyl chains. Someamphiphilic membrane anchor domains like GPI have several hydrophobicparts, several hydrophilic parts, or several of both.

Preferred membrane anchor domains are phospholipid-polyethylenglycol,stearyl, cholesterol, chelator lipid, nitrilotriacetic acidditetradecylamine (NTA-DTDA), farnesyl, myristyl or palmitoyl moieties.Most preferred are glycosylphosphatidylinositol (GPI) or analoguesthereof.

“Analogues” are mimics of GPI anchor domains wherein portions of theglycan core are systematically replaced with unnatural linkers ofcomparable length and dimension. The analogues might contain no, one ortwo mannose units and replace the phosphoinositol and glucosamine unitswith a simple hydrophilic poly(ethylene glycol) linker which allows forthe installation of various side chains and different lipid tails.Examples are given in Paulick et al (2007) which is incorporated hereinby reference.

“Target domains or target moieties” according to the present inventionare covalently joined to the hydrophilic part of membrane anchordomains. Target domains are preferably proteins or polypeptides, hereinalso named target proteins e.g. enzymes, antigen recognition sides,receptors, protein markers, fluorescence markers or proteins, or peptidehormones or cytokines. Such target domains could be further modified byjoining of protein tags and/or other functional groups (e.g. biotinylresidues, cross linkers, carbohydrates). A preferred target protein isenhanced green fluorescent protein (EGFP) or red fluorescent protein andtheir variants, especially monomeric forms (Zaccharias (2002), Campbell(2002)). CD59 or CD55 are preferred as compounds naturally consisting ofa membrane anchor domain and a target protein. CD59 or CD55 can be usedto protect viral particles against the complement system of an animal,preferably a human host. Non-proteinic target domains could be e.g.polysaccharides, nucleic acids, dyes, radioactive ligands, orfluorescent dyes. Even more complex structures like polystyrol beads or(nano)magnetic particles could be joined to a membrane anchor domain.

The term “protein tag” refers to peptide sequences genetically graftedonto a recombinant protein. Often these tags are removable by chemicalagents or by enzymatic means, such as proteolysis or splicing. Tags canbe attached to proteins for various purposes:

Affinity tags are appended to proteins so that they can be purified fromtheir crude biological source using an affinity technique. These includechitin binding protein (CBP), maltose binding protein (MBP), andglutathione-s-transferase (GST). The poly(His) tag is the mostwidely-used protein tag and it binds to metal matrices like nickel,copper and gold metal matrices. Some affinity tags have a dual role as asolubilisation agent, such as MBP and GST.

Chromatography tags are used to alter chromatographic properties of theprotein to afford different resolution across a particular separationtechnique. Often, these consist of polyanionic amino acids, such as FLAGtag.

Epitope tags are short peptide sequences which are chosen becausehigh-affinity antibodies can be reliably produced in many differentspecies. These are usually derived from viral genes and include e.g.V5-tag, c-myc-tag, and HA-tag. These tags are particularly useful forwestern blotting and immuno precipitation experiments, although theyalso find use in antibody purification.

Fluorescence tags are used to give visual readout on a protein. EGFP orred fluorescent protein and their variants are the most commonly usedfluorescence tags.

Protein tags find many other usages, such as specific enzymaticmodification (such as biotin ligase tags) and chemical modification(FLaSH) tag.

Tags can also be combined to produce multifunctional modifications ofthe protein.

Furthermore, specific moieties of tags can also be used as functionalgroups for linkage to other materials such as nanoparticles.

Tethering of target proteins to lipid membranes is achieved in severaldifferent ways, most simply by stretches of hydrophobic amino acidsforming trans-membrane domains which can be joined to proteins ofinterest. A more complex way to associate proteins to membranes is viapost-translational modification of proteins with lipophilic residuese.g. farnesyl, myristyl or palmitoyl moieties. Modification of proteinswith glycosylphosphatidylinositol (GPI) anchors probably constitutes themost complex yet most reliable way of attaching proteins to lipidmembranes.

“Glycosylphosphatidylinositol” (GPI anchor) (FIG. 3) is a glycolipidthat can be attached to the C-terminus of a protein duringpost-translational modification within a cell or chemically. It iscomposed of a hydrophobic phosphatidyl inositol group linked through acarbohydrate containing linker (glucosamine and mannose glycosidicallybound to the inositol residue) to the C-terminal amino acid of a matureprotein. The two fatty acids within the hydrophobic phosphatidylinositol group anchor the protein to the cell membrane.

GPI-anchored or “glypiated” proteins contain a signal peptide, thusdirecting them with translation into the endoplasmic reticulum (ER). TheC-terminus is composed of hydrophobic amino acids which stay inserted inthe ER membrane. The hydrophobic end is then cleaved off and replaced bythe GPI-anchor. As the protein processes through the secretory pathway,it is transferred via vesicles to the Golgi apparatus and finally to theextra cellular space where it remains attached to the exterior leafletof the cell membrane. Since the glypiation is the sole means ofattachment of such proteins to the membrane, cleavage of the group byphospholipases will result in controlled release of the protein from themembrane.

Proteins targeted for GPI anchoring contain a GPI signalling sequence(GSS) at the C-terminal end in addition to the signal peptide sequence(SP) located at the N-terminus necessary for translocation of nascentproteins into the endoplasmatic reticulum (ER). The GSS contains ahydrophilic spacer sequence of 8-12 amino acids followed by ahydrophobic region of between 8 and 20 amino acids and the site of GPIattachment is restricted to a protein specific 6 amino acids motif.Additionally, it has been shown that protein folding is not required forGPI anchor addition and hence that the size or sequence of the proteindoes not have an effect on the addition of GPI. The GSS is recognized inthe ER by the transamidase enzyme complex which consists of at least 5subunits all of which are required for correct function and is replacedby the preformed GPI anchor. The biochemical pathway for synthesis ofthe GPI anchors is complex and chemical structures of GPI anchors varyto a great degree, however a common backbone structure is observed:Linkage of the GPI anchor to the C-terminal end of the protein isachieved by an amide bond to phosphoethanolamine. The following centralthree mannose residues are linked via a non-acetylated glucosamine tothe phosphoinositol part, which in turn is associated to the lipidresidues, usually acyl or aryl fatty acid chains or sphingolipids e.g.ceramide.

A “fusion protein” according to the present invention can be produced bymethods already known in the art. These methods include, for example, invitro recombinant DNA techniques, chemical techniques and in vivorecombination/genetic recombination. DNA and RNA synthesis may,additionally, be performed using an automated synthesizer as describede.g. in Sambrook et al., (Sambrook, J, Fritsch, E F & Maniatis, T; HEds. (1989). Molecular Cloning—A Laboratory Manual, 2nd Edition. ColdSpring Habour Laboratory Press)

The preparation of such a fusion protein generally entails thepreparation of a first and second or even more DNA coding region and thefunctional ligation/joining of such regions, in frame, to prepare asingle coding region that encodes the desired fusion protein.

In a preferred embodiment, a DNA sequence coding for the target proteinis e.g. joined in frame with a DNA sequence encoding for a protein tag(TAG). The ligation can be designed for the N-terminal region or for theC-terminal region of the target protein. Depending on the proteins used,it might be necessary to introduce a peptide spacer for linkage of thetwo parts of the fusion protein. These peptide spacers could be eithercleavable or non-cleavable. The DNA sequence of the fusion protein or ofthe target protein alone is then ligated to a signal peptide sequence atthe C-terminus and a DNA sequence preferably of a GPI anchoring signalsequence (GSS) is ligated to the N-terminus.

C-terminus SP - TAG - Target protein - GSS N-terminus C-terminusSP - Target protein - TAG - GSS N-terminus C-terminusSP - Target protein - GSS N-terminusSP: Signal Peptide; TAG: Protein Tag; GSS: GPI anchoring signal sequence

Alternatively, cross-linking reagents could be used to form molecularbridges that chemically tie together functional groups of two differentmolecules, especially to join an isolated membrane anchor domain,preferably a GPI to the respective target domain.

Generally, hetero-bifunctional cross-linkers are preferred to eliminateunwanted homopolymer formation. Hetero-bifunctional cross-linkerscontain two reactive groups: one generally reacting with primary aminegroup (e.g., N-hydroxy succinimide (NHS)) and the other generallyreacting with a thiol group (e.g., pyridyl disulfide, maleimides,halogens, etc.). Through the primary amine reactive group, thecross-linker may react with the lysine residue(s) of one protein andthrough the thiol reactive group, the cross-linker, already tied up tothe first protein, reacts with the cysteine residue (free sulfhydrylgroup) of the other protein.

Therefore, polypeptides or proteins generally have, or are derivatisedto have, a functional group available for cross-linking purposes. Thisrequirement is not considered to be limiting in that a wide variety ofgroups can be used in this manner. For example, primary or secondaryamine groups, hydrazide or hydrazine groups, carboxyl, alcohol,phosphate, or alkylating groups may be used for reaction withcross-linking reagents.

The spacer arm between the two reactive groups of cross-linkers may havevarious length and chemical compositions. A longer spacer arm allows abetter flexibility of the conjugate components while some particularcomponents in the bridge (e.g., benzene group) may lend extra stabilityto the reactive group or an increased resistance of the chemical link tothe action of various aspects (e.g., disulfide bond resistant toreducing agents). The use of peptide spacers, such asL-Leu-L-Ala-L-Leu-L-Ala, is also contemplated.

It is preferred that a cross-linker having reasonable stability in bloodor other body fluids will be employed. Numerous types of disulfide-bondcontaining linkers are known that can be successfully employed.Exemplary hetero-bifunctional cross-linkers are SMPT, SPDP, LC-SPDP,Sulfo-LC-SPDP, SMCC, Sulfo-SMCC, MBS, Sulfo-MBS, STAB, Sulfo-SIAB, SMPB,Sulfo-SMPB, EDC/Sulfo-NHS or ABH. Linkers that contain a disulfide bondthat is sterically hindered may prove to give greater stability in vivo,preventing release of the agent prior to binding at the site of action.These linkers are thus one preferred group of linking agents.

One of the further preferred cross-linking reagents used is SMPT, whichis a bifunctional cross-linker containing a disulfide bond that is“sterically hindered” by an adjacent benzene ring and methyl groups. Itis believed that steric hindrance of the disulfide bond serves afunction of protecting the bond from attack by thiolate anions such asglutathione which can be present in tissues and blood, and thereby helpin preventing decoupling of the conjugate prior to the delivery of theattached viral particle to its target site e.g. a specific tissue ortumour.

The SMPT cross-linking reagent, as with many other known cross-linkingreagents, lends the ability to cross-link functional groups such as theSH of cysteine or primary amines (e.g., the epsilon amino group oflysine). Another possible type of cross-linker includes thehetero-bifunctional photoreactive phenylazides containing a cleavabledisulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)ethyl-1,3′-dithiopropionate. The N-hydroxysuccinimidyl group reacts withprimary amino groups and the phenylazide (upon photolysis) reactsnon-selectively with any amino acid residue.

In addition to hindered cross-linkers, non-hindered linkers can also beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane. The use of such cross-linkers is wellunderstood in the art.

Once conjugated, the conjugate is separated from unconjugated componentsand from other contaminants. A large number of purification techniquesknown in the art are available for use. Purification methods based uponsize separation, such as gel filtration, gel permeation or highperformance liquid chromatography will generally be of most use. Otherchromatographic techniques, such as Blue-Sepharose separation, may alsobe used.

A “pharmaceutical composition” according to the present invention mayinclude beside a therapeutically effective amount of the surfacemodified viral particle, in general, one or more pharmaceuticalacceptable and/or pharmacologically acceptable carriers, additives,antibiotics, preservatives, adjuvants, diluents and/or stabilizers.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, or ahuman, as appropriate. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. Further such auxiliary substances can be water,saline, glycerol, ethanol, wetting or emulsifying agents, pH bufferingsubstances, or the like. Suitable carriers are typically large, slowlymetabolized molecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,lipid aggregates, or the like. The use of such media and agents forpharmaceutical active substances is well known in the art. For vaccinesthe compositions may contain complementary adjuvants. For humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards or according to the EudraLex rules governingmedicinal products in the European Union.

In the present invention also procedures for modifying lipid envelopesof enveloped viruses, preferably of retroviral and lentiviral vectors(RV, LV) or herpesviral vectors with compounds which could be selectedfrom a group containing GPI protein conjugates or proteins conjugated tolipophilic moieties like ppescfpmcphospholipid-polyethyleneglycol,stearyl, cholesterol, farnesyl, palmitoyl, myristoyl, chelator lipid ornitrilotriacetic acid ditetradecylamine (NTA-DTDA) are described. Mostpreferably GPI protein conjugates are used. The virus remains infectiousafter these procedures. However, if desirable the viral vectors couldalso be inactivated prior to or subsequent to the viral paintingprocess, e.g. for vaccination purposes. Techniques are commonly known inthe art.

Potential applications include novel targeting strategies for genetherapy, immune modulation (Kueng (2007); Skountzou (2007)) (e.g.enhancement of vaccine efficacy by immune stimulation, protection ofretroviral gene therapy vectors by immune inhibition) or to implementpharmacogenetics in retroviral gene therapy by quick adaptation todifferent therapy requirements in patient sub-groups. In addition topotential clinical uses, virus painting provides a quick way tospecifically tag and modify viral envelopes e.g. for fluorescenceimaging, affinity purification, labeling to magnetic particles (seelater discussions) and radio labeling.

As model compounds CD59 (protectin, MACIF, SwissProt accession numberP13987) and EGFP linked to GPI were used. CD59 has widely been studiedand was also shown to confer partial resistance against human complementto modified murine leukemia virus (MLV) particles (Breun (1999)). Tofacilitate purification and analysis of CD59, a protein tag consistingof six consecutive histidine residues were introduced directly at theN-terminus of the mature protein. These histidine tags are alsopotentially useful in downstream applications: After painting of virionswith CD59his they can be associated via the histidine tags with e.g.magnetic nanoparticles to allow for easy purification and concentrationof virions. In addition magnetic virus could be targeted by magneticfields or used for magnetothermic therapy (Chan (2005); Ito (2005)),thus creating a new link between protein engineering andnanobiotechnology. First studies demonstrated the usefulness of magneticparticles in anti-tumor strategies (Jordan (2006), Wilhelm (2007)).

The results (see FIG. 2) show that exogenously added recombinant CD59hisassociates with concentrated retroviral and lentiviral particles. Datafrom immunoblotting suggest that the efficacy of the process allows forthe association of between 1 and 5% of the total amount of CD59his(compare FIG. 1B, 5% and 10% lanes respectively with corresponding VP+lanes) leading to estimates of between 5 and 250 molecules per virionwhich is sufficient to elicit biologically relevant effects. Dependingon the concentration of target compounds used during the incubation stepthe amount of target domains incorporated into the envelope of thepainted virus can be adjusted. It seems that there is a reciprocalcorrelation between the number of target domains anchored in the viralenvelope and the infectivity of the virus i.e sterical hindrance ofexcessive protein moieties will reduce access of the env molecules tothe cognate receptors. Sufficient results were obtained with a load of10 to 150 molecules per virion, preferably 10 to 50 molecules pervirion. Generally the infectivity was slightly reduced when compared toinfection levels before painting. This could be due to a sterichindrance of the natural viral envelope proteins. However, whencomparing infection levels for GPI-associated virions to non-associatedvirus particles only a small difference is observed (FIG. 2, comparelanes VP− and VP+). This indicates that the duration of the process (5to 6 hours in total) rather than the painting process itself isresponsible for the decrease in infectivity. Using shorter incubationtimes for painting and alternative purification post-painting procedurese.g. ultrafiltration or magnetic purification decrease handling timesleads to higher infection rates.

As the vast majority of proteins can be removed by post-paintingprocedures like e.g. ultracentrifugation, the incorporation processappears to be specific. The control protein (rat IgG) is a solubleprotein of hydrophilic character with consequentially low affinity forhydrophobic surfaces. The possibility remains that proteins withsubstantial hydrophobic sequence motifs, e.g. trans-membrane proteins,could show comparable behaviour to GPI proteins, somehow similar to theformation of proteoliposomes. Virions remained infectious after paintingand post-painting treatments (FIG. 2).

Another advantage of using GPI anchors for modification of proteins isthat the amino acid sequence of the mature protein is not alteredincreasing the possibility for functionally intact proteins. Addition ofthe hydrophobic moiety might however influence folding of therecombinant GPI protein thereby destroying conformational epitopes orthe active centers of enzymes. Moreover, preliminary experimentssuggested that levels of painting are increasing with increases of virusnumbers and GPI protein amount. This indicates that the amount of GPIproteins after painting on virions is controllable. There are a numberof potential uses for this technique: Addition of immune-stimulatoryGPI-linked factors could enhance the efficacy of vaccination (Skountzou(2007)) or provide an important added benefit for viral suicide genetherapy. Immune inhibitory molecules might help to protect viral genetransfer vectors from unwanted or premature immune responses. Theinherent flexibility of a modular system (i.e. adding different types ofGPI-linked molecules to viruses directly prior to use) would allow forquick and adaptive responses to therapy needs for example in response togenetic heterogeneity in patients.

Virus painting can also be used to attenuate existing or new vaccines toenhance safety by either (i) reducing virulence or the efficiency of theinfection event (ii) targeting the virus so that it preferentiallyinfects cells that do not allow productive infection or (iii)retargeting to professional antigen presenting cells e.g. targeting toC-type lectin receptors is known to induce potent helper and cytolyticT-cell responses (Keler (2007)) or e.g. using a chimeric ErbB2 linked toCTLA-4 B7 interacting domain (Rohrbach (2005)).

Viral painting also allows for the direct labeling of viral envelopemembranes, thus facilitating concentration, purification and/orvisualization of virions e.g. for diagnostic purposes. Incorporation ofspecific chemical moieties into viral envelops are also useful forlowering of detection limits in common qualitative and quantitativeimmunoassays, such as ELISA or for PCR and/or FACS analysis. The methodspresented herein are especially useful as part of a diagnostics systemor diagnostic kit. Furthermore they can be used for isolation,detection, or measurement of known or unknown enveloped virus.

GPI tagging allows for specific concentration of known or unknownenveloped virus in any given sample, preferably in body fluid, cellculture medium or buffered saline. Therefore, new applications for viruspurification and/or concentration and/or isolation are presented. Thistool may conceivably developed as a major component of a new diagnosticssystem to quickly and easily purify and identify and/or quantify knownor unknown viruses from biological samples, research samples or clinicalsamples. Currently, classic ultra-centrifugation techniques or moremodern ultra-filtration methods using regenerated cellulose, poly ethersulphone or cellulose triacetate commercially available membranes fromSartorius (Vivaspin) or Millipore (Centricon) are used tonon-specifically collect or concentrate viruses from samples. Thesemethods are however non-specific, i.e. all viruses and everything havingthe same size as viruses will also be collected e.g. serum proteincomplexes from cell culture media. This can lead to many diverseproblems with downstream protocols due to such contaminations. Alsoultra-filtration only works in certain settings due to problems withnon-specific sticking of virus with the membranes. Hence a specific wayto tag enveloped viruses from a sample via compounds, preferablyrecombinant GPI proteins with histidine tags and magnetic nanoparticleswould be a novel and improved way to achieve purification for furtherdownstream identification methods such as ELISA, or PCR based assays.FIG. 1 shows that the GPI protein associates to retroviral andlentiviral vectors and FIG. 5 shows exemplarily association with otherenveloped virus types, in this case feline herpes virus. FIG. 6 showsthat 2 model recombinant GPI proteins (namely CD59 and GFP) can both behighly efficiently attached to and manipulated by magnetic force byvirus-sized magnetic nanoparticles (MNP). This clearly shows that bothnecessary components of the proposed system are functional, i.e.GPI-virus and GPI-MNP interactions. An additional benefit is thatinfection based assays, e.g. focus forming unit or plaque assays canalso be adopted as the GPI tagging process does not affect the virusinfectivity in contrast to the more conventional methods describedabove. For completeness, it must also be mentioned that affinitytechniques, mostly based on column format using anti-viral envelopeprotein antibodies, is also an option for specifically isolatingviruses, however, this is limited to a situation where the virus isknown, it is expensive and also leads to the purification of anon-infectious virus and is therefore also not viable alternative to theapplications suggested above for newly proposed GPI tagging tool.

We have also successfully demonstrated that the methods also work withother enveloped viruses such as influenza virus, pox virus, HBV andherpes virus, for an example see feline herpes virus (FIG. 5). Theprotein tags were engineered into CD59his initially for the purpose ofeasy purification. However, the tags can also be used for the linkage ofmagnetic micro- or nanoparticles to the CD59his and subsequently to thevirion, thus creating a link between retrovirology and nanotechnology.Magnetic retrovirus could be easily concentrated and purified viaexposure to magnetic fields (Chan (2005)). In addition magnetic fieldscould be used in targeting particles and inducing hyperthermia (Ito(2005); Jordan (2006); Wilhelm (2007)). Taken together, the directtransfer of GPI-linked his-tagged proteins onto enveloped viralparticles is a potentially valuable tool for a variety of both researchand clinical applications.

SUMMARY OF THE INVENTION

A method for exogenously modifying the envelope composition of anenveloped viral particle, comprising the steps

(a) Concentration of isolated viral particles from a suspension fluid

(b) Incubation of the concentrated viral particles with a reactantconsisting of a hydrophilic target domain or moiety covalently linked toa lipophilic membrane anchor domain or moiety, wherein the lipophilicmembrane anchor domain becomes integrated into the lipid double layer ofthe virus envelope and wherein the hydrophilic target domain becomesexposed to the surrounding watery incubation fluid(c) Separation of envelope modified viral particles from excessivereactants

A method comprising the steps

(a) Incubation of a fluid containing enveloped viral particle with areactant consisting of a hydrophilic target domain or moiety covalentlylinked to a lipophilic membrane anchor domain or moiety, wherein thelipophilic membrane anchor domain becomes integrated into the lipiddouble layer of the virus envelope and wherein the hydrophilic targetdomain or moiety becomes exposed to the surrounding fluid(b) Separation of envelope modified viral particles from excessivereactants(c) Detection of the envelope modified viral particles.

Methods as described above, wherein the total protein content of theviral envelope in relation to the total lipid content of the viralenvelope is between 50:50 and 90:10, preferably 65:35 to 85:15 and mostpreferably 70:30 to 80:20 mol %

A method as above wherein the lipophilic membrane anchor domain isselected from the group comprising phospholipid-polyethyleneglycol,stearyl, myristyl, cholesterol, chelator lipid nitrilotriacetic acidditetradecylamine (NTA-DTDA) and glycosylphosphatidylinositol (GPI) oranalogues thereof, whereas analogues of GPI anchor domains are mimicswherein portions of the glycan core are systematically replaced withunnatural linkers of comparable length and dimension. Those analoguesmight contain no, one or two mannose units and replace thephosphoinositol and glucosamine units with a simple hydrophilicpoly(ethylene glycol) linker which allows for the installation ofvarious side chains and different lipid tails

A method as above wherein the hydrophilic target domain is selected fromthe group comprising polysaccharides, nucleic acids, dyes, radioactiveligands, fluorescent dyes, synthetic beads or magnetic particles,wherein the synthetic beads or the magnetic particles can be coated oruncoated.

A method as above, wherein the hydrophilic target domain is a protein ora polypeptide.

A method as above, wherein the protein or the polypeptide furthercontains a protein tag or combinations of protein tags.

A method as above, wherein the protein or the polypeptide is an enzyme,an antibody, a receptor, a marker protein, a fluorescence protein, acomplement inhibitor or a cytokine.

A method as above wherein the complement inhibitor is CD59 or CD55 andthe cytokine is a interleukin and/or colony-stimulating factor,preferably IL-2, IL-4 or IL-12

A method as above, wherein the hydrophilic target domain and thelipophilic membrane anchor domain are chemically joined by a crosslinker.

A method as above, wherein one or more different reactants are used orcombined.

A method as above, wherein the suspension fluid is a body fluid, a cellculture medium, a physiological saline or a buffered saline

A method as above, wherein the concentration of isolated viral particlesand/or separation of envelope modified viral particles is achieved byultracentrifugation, ultrafiltration or chromatography, especiallyaffinity chromatography

A method as above, wherein the incubation step is achieved by slowagitation in body fluid, cell culture medium, buffered saline orphysiological saline at temperatures between 4 to 40° C. for incubationtimes between 10 min and 48 hours

A method as above, wherein the temperature is preferably between 25 and38° C. and/or the incubation time is between 1 and 3 hours.

A method as above, wherein the enveloped viral particle is selected fromthe group comprising Arenaviridae, Bunyaviridae, Coronaviridae,Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae,Orthomyxoviridae, Paramyxoviridae, Poxyiridae, Retroviridae,Rhabdoviridae and Togaviridae

A method as above, wherein the viral particle is a retrovirus,especially mouse leukemia virus or a lentivirus or a poxvirus,especially small poxvirus or a vaccinia virus preferably MVA or NYVAC ora herpesvirus, especially feline herpes virus.

A method as above, wherein the genome of the viral particle isgenetically modified compared to its wild-type form.

A method as above, wherein the viral particle is inactivated prior to orsubsequent to the painting method.

A method as above, wherein the painting process is carried out prior to,simultaneously with or post to other methods for modifying the viralenvelope.

A viral particle produced by a method as above.

A pharmaceutical composition containing a therapeutically effectiveamount of the viral particle as above and a pharmaceutically acceptablecarrier

Use of the viral particle or of the pharmaceutical composition as aboveas a medicament

Use of the viral particle or of the pharmaceutical composition as abovefor gene therapy, vaccination, or immunomodulation

Use of the viral particle as above for attenuation of vaccinesespecially to enhance the safety of vaccines by (i) reducing virulenceor the efficiency of the infection event (ii) targeting the virus sothat it preferentially infects cells that do not allow productiveinfection and/or (iii) retargeting to professional antigen presentingcells e.g. targeting to C-type lectin receptors.

Use of the method as above for concentration of viral particles as anenrichment tool for research and diagnosis purposes, especially tofacilitate said research/diagnostic applications by removal ofcontaminants (e.g. salts, proteins and protein complexes).

A diagnostic method as above, wherein the enveloped viral particles aredetected by Immunoassays, chromatography, FACS analysis or microscopy.

Use of the method as above for diagnosis and/or visualization of virusparticles.

A kit containing at least the reactant containing a hydrophilic targetdomain or moiety covalently linked to a lipophilic membrane anchordomain or moiety for use in the methods described above, andinstructions for performing the methods as above.

SHORT DESCRIPTION OF THE DRAWINGS

FIG. 1: Painting of retroviral and lentiviral vector particles. (a)Overview: concentrated supernatants from retroviral (RV) or lentiviral(LV) producer cell lines (293 gpalfpLXSNeGFP and STAR-A-HV,respectively) are incubated with purified and concentrated CD59his for3-20 hours at 37° C. under constant shaking. After incubation samplesare purified by ultracentrifugation (2 hrs, 20 000 rpm, 4° C.) to removenon-virus-associated proteins. Before analysis of CD59his, endogenousCD59 is removed by using magnetic nickel beads (Promega). Samples wereanalysed by immunoblotting using antibodies directed specificallyagainst CD59, MLV capsid (CA) and HIV-1 p24. (b) Analysis of paintedretrovirus: concentrated supernatants from parental cells (PC) and virusproducing cells (VP) were incubated in the presence or absence ofCD59his for 21 hours at 37° C. under constant shaking. In addition cellculture medium (ME) was also incubated under the same conditions. Afterpurification as detailed above, cells were analysed by immunoblotting.Results show that CD59his is only retained during purification in thepresence of virus and CD59his (RV and LV, upper panels respectively,compare lanes VP− and VP+) indicating association of the protein withviral particles. On the same gels, either 5 or 10% respectively of theamount of CD59his used for viral painting was loaded to assessefficiency of painting. Levels of viral gag proteins are shown viaimmunoblots using MLV capsid and HIV-1 p24 antibodies. Viral proteinsare only present in supernatants derived from viral producers. (RV andLV, lower panels respectively, compare lanes VP− and +). (c) Specificityof viral painting: concentrated viral supernatant were mixed withCD59his and the same amount of a non-GPI protein (rat IgG; MW 150 kD;Dako). The sample was incubated for approximately 20 hours. Aliquots aretaken before and after ultracentrifugation and silver stained to assessprotein content 100% and 10% of the used amount of IgG were loaded forcomparison. Ultracentrifugation removes the majority of proteins as wellas the IgG contaminant. (d) Infectivity after Painting: Virussupernatants post-painting are purified by ultracentrifugation and usedto infect target HeLa cells. After 36 hours supernatant is removed andanalysed by immunoblotting for presence of CD59his to confirm painting.Cells analysed by flow cytometry. No infection was observed in HeLacells treated with medium (mock) or supernatant from parental cells thatwas incubated with CD59his (PC+). Virus supernatant in the absence orpresence of CD59his (VP−; VP+) shows infection rates of approximately4%. Supernatants treated with CD59his performed slightly worse ininfection experiments. As infection control viral supernatant savedbefore painting (one tenth of the volume used for infection afterpainting) was used (VP). Infectivity was reduced significantly afterpainting. Error bars represent means+/−standard deviation.

FIG. 2: Glycosylphosphatidylinositol (GPI) anchors. (a) The N-terminalsignal peptide (SP) of the nascent polypeptide chain leads totranslocation to the ER. The transamidase complex located at the ERmembrane, recognises the GPI anchoring signal sequence (GSS) andreplaces it with the preformed GPI anchor. (b) A common backbonestructure is observed in GPI anchors. Linkage to the protein part isachieved by an amide bond to phosphoethanolamine. The followingcarbohydrate core consist of three mannose and a glucosamine residue.The phosphoinositol group links the hydrophobic lipid part, mainly aryl,acyl or ceramide type lipids, to the remaining GPI anchor. Thehydrophobic moieties are inserted into the outer leaflet of the cellmembrane. Sites of cleavage for mammalian GPI-specific phospholipases Cand D (GPI-PLC and GPI-PLD, respectively) are indicated by arrows.

FIG. 3: Serum protection after viral painting. An experiment (n=1) wascarried out to determine serum protection of virions painted withCD59his. After painting incubation and post-painting purification stepspainted viral particles were challenged with human serum for 1 hour at27° C., as were unpainted viral particles. 48 hours post infection thetarget HeLa cells (ATCC No. CCL-2) were analysed for infection by flowcytometry. Infectivity of unpainted viral particles was reduced to about16% after challenging with human serum, compared to not challenged viralparticles. The observed infectivity loss in painted viral particles wasless pronounced (to 60%), as expected if serum protection from CD59histakes place.

FIG. 4: Varying amounts of GPI-anchored proteins and viral particles.Varying amounts of CD59his (1×, 4×, 16× relative amounts) were incubatedwith the same amount of viral particles (2× relative amount). Afterpost-painting purification steps immunoblots for CD59 show moreassociation of CD59his to viral proteins when higher concentrations ofCD59his were used as starting material. When the same concentration ofCD59his was used to paint different amounts of viral particles (1×, 2×,4× relative amounts) a medium concentration of viral particles seemed toperform best (2× relative amount).

FIG. 5: Painting of feline herpesvirus 1 (FHV-1):

Concentrated supernatants from CrFK cells infected with FHV-1 or treatedwith hygromycin were used in a painting experiment, along with cellculture medium—incubation was carried out in the presence or absence ofCD59his for 20 hours before post-painting purification byultracentrifugation. A) Immunoblot using specific antibodies directedagainst CD59 were carried out. Signals can be detected for samplesincubated in the presence of CD59 from supernatants from infected(CrFK-FHV+) and—to a lesser degree—hygromycin-treated cells (lanesCrFKHyg+). To determine activity of virus post-painting, aliquots ofsamples were used to infect CrFK cells. The cell destruction orcytopathic effect (CPE) was analysed 24 hours post infection by phasecontrast light microscopy. Strong CPE was detected in samples containingFHV-1 particles only (CrFK-FHV− and +, respectively). This indicatesthat virus remains infectious during the procedure. B) Cytopathiceffects (CPE) upon infection of CrFK cells with painted FHV-1 particles.In the presence of complete, biologically active virus CrFK cells areinfected and damaged (see lane FHV). After painting CPE can be detectedin cells infected with the CrFK-FHV samples only, confirming presence ofactive virus post-painting (see lanes CrFK-FHV− and +). A confluentmonolayer is observed in samples not containing viral particles(Medium−/+; CrFK−/+).

FIG. 6: Painting with GPI-anchored green fluorescent protein.

Lentiviral particles derived from STAR producer cells were incubatedwith GPI-anchored monomeric green fluorescent protein (mGFP-GPI).Post-painting samples were purified by ultracentrifugation and analysedby immunoblotting for presence of mGFP-GPI (upper panel) and p24 (lowerpanel). mGFP-GPI was only present when viral particles were present, andthe sample had been treated by painting with mGFP-GPI (lane VP+) and toa lesser extent when supernatant from non-virus producing parental cellswas treated by painting (lane PC+) due to the presence of lipid vesiclesof non-viral origin.

FIG. 7: Magnetic nanoparticles (MNP) associate specifically withrecombinant GPI proteins

GPI-anchored green fluorescent protein was mixed with magneticnanoparticles (Nickel-NTA-coated iron core particles, average size 5-10nm). MNPs associated with the target proteins could be retained duringthree washing procedures after magnetic manipulation (lane B). Most ofcellular protein was lost (compare lanes B and A, bottom panel,Coomassie-stained gel), whereas a comparatively large proportion of thetarget protein stayed associated throughout the process (compare lanes Band A, top panel, immunoblot for GFP). This indicates that magneticmanipulation of GPI-anchored proteins is possible when using magneticnanoparticles. Lane M exhibits the molecular weight marker.

EXAMPLES Example 1 Production of CD59his

CrFKCD59hisneo cells expressing the recombinant CD59his were derivedfrom parental CrFK cells by lipofection using lipofectin reagentaccording to manufacturer's instructions (Invitrogen) with pCD59hisneo.For generation of pCD59hisneo a PCR fragment derived from cDNA (usingprimers CD59(2)FKHindIII 5′-cacgacaagcttaccatgggaatccaaggawgtctgtcctgtt-3 SEQ ID No: 5) andCD59(2)RApaI5′-atgacgggcccttagggatgaaggctccaggctgctgccagaa-3′ SEQ ID No:6) from HEK293 cells was cloned into the expression vector pcDNA3(Invitrogen). The his-tag was introduced by a two-step mutagenesis PCRprotocol, using first two primer pairs (CD59(2)FKHindIII & CD59RHis5′-gtgatggtgatggtgatggctatgacctgaatggcagaag-3′ SEQ ID No: 8; CD59FHis5′-catcaccatcaccatcacctgcagtgctacaactgtccta-3′ SEQ ID No: 7 andCD59(2)RApaI) in two different PCR reactions. Subsequently a mix of bothprimary fragments was hybridized and amplified using primersCD59(2)FKHindIII and CD59(2)RApaI. The fragment was recloned into pcDNA3using the HindIII and ApaI sites. 293 gpalfpLXSNeGFP are derived fromHEK293 cells (Ikeda (2003); Klein (1997); Pambalk (2002)). STAR-A-HV(Wilhelm (2007)) are derived from HEK293T cells).

Example 2 Purification of CD59his

4-6 confluent T175 flasks of CrFKCD59hisneo were harvested by scrapingafter washing cells with 10 ml PBS. Cells were scraped into a total of25 ml sample application buffer (50 mM TrisHCl, 50 mM NaCl, 35 mMImidazole, 0.5% sodium deoxycholate, 1% NP40, pH 7.4). 80 μl of proteaseinhibitor complex (Sigma) was added before sonification of samples for30 seconds. Samples were incubated for 30 minutes on ice beforecentrifugation for 30 minutes at 2000 g. Samples were filtered through0.2 μm filters (Sarstedt) before application to a ÄktaPrime plus FPLCdevice (GE Healthcare). Prepacked 5 ml HisTrap FF Crude columns (GEHealthCare) were used. Samples were washed using washing buffer (50 mMTrisHCl, 50 mM NaCl, 35 mM Imidazole, pH 7.4) and eluted from columns byelution buffer (50 mM TrisHCl, 50 mM NaCl, 600 mM Imidazole, pH 7.4).Fractions were collected during elution. Presence of CD59his infractions was determined by immunoblotting. Positive fractions werepooled and concentrated by ultrafiltration using Amicon Ultra filterdevices (Millipore, 5 kD molecular weight cut-off). Samples were washedtwice with 5 ml painting buffer (50 mM TrisHCl, 50 mM NaCl, pH 7.4).Concentrations were measured using the DC protein assay (BioRad).

Example 3 Painting of Virus with CD59his

Supernatants from the stable lentiviral producer cell line STAR-A-HV(Ikeda (2003)) or the MLV-based retroviral producer cell line 293gpalfpLXSNeGFP (Klein (1997); Pambalk (2002); Hlavaty (2004)) wereharvested, filtrated through 0.45 μm filters (Sarstedt) and viralparticles were concentrated by ultracentrifugation (2 hrs, 20 000 rpm,4° C.) in a Beckmann XL-70 ultracentrifuge using a SW28 rotor andresuspended in DMEM cell culture medium (Gibco), before incubation withCD59his at final concentrations between 20 and 100 ng/μl for 21-24 hoursat 37° C. and 5% CO₂. For painting, supernatants derived fromconcentration of 2-6 T175 culture flasks were incubated with purifiedprotein at final concentrations between 20 and 100 ng/μl or paintingbuffer alone. Incubation was carried out at 37° C., 5% CO2 underconstant shaking. Incubation times were 3 (infection experiments) toapproximately 21 hours (standard experiments). To separate potentiallypainted virus from free GPI-linked proteins, samples were diluted byaddition of 34 ml of DMEM and ultra-centrifuged (2 hrs, 20 000 rpm, 4°C.). To allow for the differentiation between recombinant CD59his andendogenous CD59 present on virus producer cells, samples were subjectedto purification with Ni-magnetic particles (MagneHis kit, Promega) afterpainting according to the instructions of the supplier andultracentrifugation to remove endogenous CD59 derived from producercells (for an overview of procedure see FIG. 1A). CD59his was detectedonly in samples containing virus and purified CD59his, suggesting thatboth constituents are necessary (FIG. 1B, lane VP+). No influence onpainting was observed as a result of the used media (FIG. 1B, lane ME−)or the parental (non-virus producing) cells (FIG. 1B, lane PC+) used.However, if cells undergo considerable stress i.e. overgrowing inculture prominent amounts of non-viral membrane vesicles can be shed(Taraboletti (2006)) leading to the potential for painting of theseexosomal bodies as well. In addition, post-painting procedures weresufficient to remove unpainted CD59his (seen by the absence of a signalfor CD59his in the ME+ sample, FIG. 1B) as well as endogenous CD59 (seenby the absence of a signal for CD59his in the PC− sample, FIG. 1B).

Proteins may however stick to viral envelopes regardless ofGPI-anchoring in a non-specific manner. Silver staining of paintedsamples before and after purification via ultra-centrifugation showedthat the majority of proteins are removed in the purification step (FIG.1C). In addition, we added rat IgG at the same levels as CD59his to thepainting reaction. IgG was not retained by the virus as the CD59his was(FIG. 1C). This indicates that the process is at least semi-specific.Potentially proteins with pronounced hydrophobic stretches e.g.trans-membrane proteins could interact in a way similar to GPI proteinswith lipid membranes.

Optimisation was carried out to determine the minimal incubation timenecessary for membrane re-insertion. Preliminary results suggested thatan incubation time of 3 hours is sufficient for maximal viral painting.Using the minimal incubation time, painting experiments were repeated,to assess infectivity of painted virus. HeLa (ATCC No. CCL-2) cells areinfected with painted virions and analysed by flow cytometry 36 hourspost infection. Supernatant after infection was collected and analysedfor CD59his to confirm painting. (FIG. 2). Painted virus remainsinfectious, however at reduced levels. Differences in infectivitybetween samples that received CD59his and mock-painted samples that didnot receive GPI proteins are small (FIG. 2, compare samples HV− andHV+). The difference of infectivity to samples before painting wascomparatively large (approximately 15-fold, FIG. 2, compare samples HV,HV− and HV+). This indicates that the reduction in infectivity is rathera result of the duration of the process than the process itself.

Example 4 Painting Stoichiometry

Calculations of the stoichiometry of the viral painting process,especially the numbers of GPI proteins incorporated per virus are basedon viral titers determined by product enhanced reverse transcriptase(PERT) and by determination of viral painting efficacy from immunoblots.The density of CD59 per virion is defined as the number of totalassociated molecules N_(MA) divided by the number of virions N_(V),determined by product enhanced reverse transcriptase (PERT) assay. ThePERT assay was carried out as described in (Steinrigl (2007)). Beforeelectroblotting (1.1 mA/cm²) onto PVDF membranes (Hybond P, GEHealthCare), samples were electrophoretically separated on pre-cast4-12% gradient gels (NuPage, Invitrogen). Monoclonal antiCD59 waspurchased from Serotec. Mouse anti human HIV-1 p24 was purchased fromPolymun Scientific (Vienna). MLV anti capsid antibody was purified byBiomedica. HRP-conjugated anti-rat and anti-mouse secondary antibodieswere purchased from DakoCytomation. Signal detection was carried outusing the ECLplus kit (GE HealthCare)

The density (D) of CD59his molecules per virion is dependent on theamount of CD59his (M [g]), the efficacy of the association process(E_(A)) and the number of virions (N_(v)), determined by productenhanced reverse transcriptase (PERT) assay. The constant factor kcontains the parameters supposed to not change between experiments, suchas the molecular weight (M_(w)) of the GPI protein (20 kDa), theefficacy of purification (E_(r)) and the Avogadro number (N_(a)).Following formula can be used for calculation of the stoichiometry:k=(E _(P) ×N _(a)/(M _(w)×10E9);D=k×(M×E _(A))/N _(v)Results for the experiments depicted in FIG. 1B suggested that between 5and 250 molecules can be found per virus. Experiments carried out usingeither the same concentration of CD59his on varying viral concentrationsor vice versa showed that the amount of incorporation of CD59his intoviral envelopes is dependent on viral titers and CD59his concentration(see FIG. 4), whereas the number of inserted CD59his molecules increasewith increased amounts of CD59his. In parallel the infectivity of theviral particles decrease with increased numbers of inserted CD59his dueto a steric hindrance of natural viral envelope proteins. The bestrelation between the number inserted compounds and infectivity can beachieved with 50 to 150 CD59his molecules per virion.

Example 5 Infection of Hela Cells and Flow Cytometry

For infection 8-9×10⁵ HeLa target cells (ATCC No. CCL-2) were seeded 6hours prior to infection in 6 well plates. Virus supernatants afterpost-painting ultracentrifugation were diluted to 1 ml with DMEMsupplemented with 10% FCS (Gibco) and 10 μl/ml polybrene (0.8 μg/μl).After 36 hours Supernatants were saved for analysis of CD59his content.Cells were trypsinised, fixed, washed 2 times in PBS and analysed forexpression of eGFP in a FACsCalibur flow cytometer (BectonDickinson)using CellQuest software.

Example 6 Painting of Feline Herpesvirus 1 (FHV-1)

Crandell feline kidney cells (CrFK, ATCC No. CCL-94) were infected withFHV-1 (2 ml concentrated suspension per T175 flask) and incubated untilcomplete destruction of cells took place (approximately 48 hours). Inparallel, the same amount of CrFK cells was treated with hygromycin(Invitrogen, 200 μg/ml final concentration) to simulate the cell damageusually associated with FHV infection. The supernatants were harvestedby ultracentrifugation (2 hrs, 20K rpm, 4° C. SW28 rotors, using anBeckman XL-70 ultracentrifuge) 48 hours post infection and resuspendedin DMEM w/o FCS (Invitrogen). Both concentrated supernatants as well asthe same amount of just DMEM w/o FCS were incubated for 20 hours in thepresence or absence of purified CD59his (Final concentration up to 100ng/μl, see example 1 and 2 for production and purification of CD59his))at 37° C. under constant shaking. Viral particles were separated fromnot associated CD59his by ultracentrifugation as described above. Thesamples were then resuspended in DMEM w/o FCS post ultracentrifugationand aliquots used for immunoblotting (to assess association of CD59 toviral particles) or infecting confluent layers of CrFK cells kept inDMEM w/o FCS (to assess presence of viral particles post-painting bydetermining the cytopathic effect—CPE).

Example 7 Production of mGFP-GPI

To achieve expression of mGFP-GPI the sequence coding for the monomericGFP as described by Zacharias et al (Zacharias (2002) was cloned into avector backbone (pcDNA3.1hyg+ (Invitrogen)) providing the his-tag andthe GSS of human decay accelerating factor (DAF, CD55) in a 2 stepmutational PCR protocol, similar to the one explained in example 1. Toprimer sets were used: MEHindIIIF(5′-cgcgcgcaagcttaatcaaaacatggctcagcggatgaca-3′) SEQ ID No: 1 andMonoHisEG3R (5′-gtggtggtgatggtggtgcttgtacagacgcccatgccgagagt-3′) SEQ IDNo: 2 in the first set; HisEG1F(5′-caccaccatcaccaccacccaaataaaggaagtggaacc-3′) SEQ ID No: 3 and EGApaIR(5′-gaatagggccctaagtcagcaagcccatg-3′) SEQ ID No: 4 in a second set.Primers MEHindIIIF and EGApaIR were then used to amplify the completesequence. The fragment was cloned into pcDNA3.1hyg+(Invitrogen) usingthe unique HindIII and ApaI sites. Transfection of HEK293 cells wascarried out as described in example 1. Purification and concentration ofmGFP-GPI were carried out as described in example 2.

Example 8 Painting with Green Fluorescent Protein (GFP) Variant Proteins

Viral particles were harvested from STAR cells (Ikeda et al. (2003)) asdescribed previously (see example 3). Proteins were purified andconcentrated as described previously (see example 2). Cell culturesupernatants were concentrated as described previously (see example 3).Purified proteins were incubated with supernatant derived from 4 T175flasks per sample at final concentrations up to 100 ng/μl protein.Painting reaction was allowed to commence for 20 hours at 37° C. underconstant shaking before ultracentrifugation (as described previously,example 3). No magnetic pre-purification was necessary, as no endogenousGFP can contaminate the samples.

Example 9 Detection of CD59 and mGFP-GPI

Samples were separated on precast 4-12% gradient gels (Invitrogen) undernon-denaturing conditions in MES buffer at 100 V. Electroblotting ontoPVDF membranes (GE Healthcare) was carried out at 1.1 mA/cm2 for 1 hour.Membranes were blocked overnight in 4% milk powder and 1% bovine serumalbumin (Sigma-Aldrich) dissolved in TTBS (5% v/v Tween 20, 150 mM NaCl,20 mM TrisHCl pH 8.0). Primary antibodies for CD59 (Serotec), p24(Polymun) and EGFP (Invitrogen) were used at dilutions of 1:2000 and1:1000 (EGFP), respectively. Secondary antibodies conjugated to horseradish peroxidase (DakoCytomation) against mouse and rabbit IgG wereused at dilutions between 1:5000 and 1:10 000. Signal detection wascarried out using the ECLplus kit (GE HealthCare)

Example 10 Silver Staining of Proteins

Silver staining of protein extracts was carried out as previouslydescribed (Shevchenko et al. (1996). In brief: After fixing and washing,the polyacrylamide gels were sensitized in a 0.02% sodium thiosulfatesolution for 1 minute. An aqueous 0.1% silver solution was used for theincubation before development in a sodium carbonate/formaldehydesolution. Color development was stopped by washing in 5% acetic acid inwater.

Example 11 Magnetic Nanoparticles (MNP) Associate Specifically withRecombinant GPI Proteins and Allow Magnetic Manipulation

GPI-anchored 6× histidine tagged green fluorescent protein or GPIanchored 6× histidine tagged CD59 was expressed in HEK293 as describedpreviously (see examples 1 and 7). In brief after two-step mutagenesisPCR to introduce the 6×His tag resulting plasmids were transfected intoHEK293 cells by lipofection (Invitrogen). Total cell extracts fromexpressing cells were mixed with iron based, phospholipid micellenickel-nitrilo-acetate coated MNPs (Lim (2006); size of 5-10 nm or 50 nmdiameter). For binding to target proteins and isolation, MNPs are addedto total protein lysates after sonication and mixed for 4 hours at roomtemperature, then placed into a magnetic stand (Qiagen) and supernatantcollected for further testing. Particles plus protein pellet is washedwith wash buffer containing 1 mM Imidazole in 1× extraction Buffer(0.15M NaCl, 0.05 M Tris pH 7.5, 1% v/v NP40 (Sigma), 0.5% w/vSodiumdeoxycholate (Sigma) and mixed by pipeting. This process isrepeated twice so that three washing steps are performed in total. Boundprotein-MNP can be then used for painting experiments or eluted usinghigh concentrations of imidazole (500 mM) (and hence purified forfurther analysis). Cells were analysed by immunoblots using GFP specificantibodies (Invitrogen) and Coomassie staining of polyacrylamide gels.Levels of cellular protein are dramatically reduced by the purificationstep (as can be seen in the Coomassie staining, FIG. 7, bottom panel)and a large portion of the total amount target protein is recoveredafter purification (FIG. 7 lane A), when compared with the completeextract (FIG. 7, lane B)

Primer used MEHindIIIF (5′-cgcgcgcaagcttaatcaaaacatggctc agcggatgaca-3′)SEQ ID No: 1 MonoHisEG3R (5′-gtggtggtgatggtggtgcttgtacagctcgtccatgccgagagt-3′) SEQ ID No: 2 HisEG1F(5′-caccaccatcaccaccacccaaataaagg aagtggaacc-3′) SEQ ID No: 3 EGApaIR(5′-gaatagggccctaagtcagcaagcccatg-3′) SEQ ID No: 4 D59(2)FK(5′-cacgacaagcttaccatgggaatccaagg HindIII agggtctgtcctgtt-3)SEQ ID No: 5 CD59(2)RApaI (5′-atgacgggcccttagggatgaaggctccaggctgctgccagaa-3′) SEQ ID No: 6 CD59FHis(5′-catcaccatcaccatcacctgcagtgcta caactgtccta-3′) SEQ ID No: 7 CD59RHis(5′-gtgatggtgatggtgatggctatgacctg aatggcagaag-3′) SEQ ID No: 8

REFERENCES

-   Beer C. et al. Virol J 22, 36-44 (2005)-   Breun, S., et al. BBRC 264, 1-5 (1999).-   Brügger, B. et al. Retrovirol 4, 70-82 (2007)-   Campbell R E. PNAS 99, 7877-7882 (2002)-   Chan, L., et al. J. Virol 79(20), 13190-13194 (2005).-   Hlavaty, J., et al. J. Virol 78(3), 1384-1392 (2004).-   Ikeda, Y., et al. Nature Biotechnology 21, 569-572 (2003).-   Ito, A., et al. J. Bioscience and Bioengineering 100 (1), 1-11    (2005)-   Jordan, A., et al. J Neuro-Oncology 78, 7-14 (2006)-   Keler, T, et al., Oncogene 26 3758-3767 (2007),-   Klein, D. et al. Gene Therapy 44, 1256-1260 (1997).-   Kueng, H J et al. J. J. Virol. 81(16), 8666-8676 (2007).-   Legler, D. F., et al FASEB J. 19, 73-75 (2005).-   Lim Y T et al. Biochem Biophys Res Commun 344 926-30 (2006)-   McHugh, R. S. Proceed. Natl. Acad. Sci. USA 92, 8059-8063 (1995).-   Medof, M. E et al., FASEB J. 10, 574-586 (1996).-   Metzner et al. FASEB J. 22, 2734-2739 (2008)-   Metzner et al. Virol 382, 125-131 (2008)-   Morandat, S., et al. Biochim. Biophys. Acta 1564(2), 473-478 (2002).-   Pambalk, K., et al. BBRC 293, 239-246 (2002).-   Paulick, M G et al. J. Am. Chem. Soc., 129:11543-11550 (2007)-   Premkumar, D. R. D.; et al. J. Cell. Biochem. 82, 234-245 (2001).-   Rohrbach, F et al., 2005, J. Immunol. 174 5481-5489 (2005).    174:5481-9).-   Ronzon, F., et al J. Membr. Biol. 197(3), 169-177 (2004).-   Roux, P et al. Proc. Natl. Acad. Sci. 86, 9079-9083 (1989)-   Schevchenko, A et al. Anal. Chem. 68, 850-858 (1996)-   Skountzou, I., et al J. Virol. 81(3), 1083-1093 (2007).-   Steinrigl, A., et al. Virology 362(1), 50-9 (2007)-   Taraboletti, G., et al. Neoplasia 8(2) 96-103 (2006).-   Wilhelm, C et al. J Nanosci Nanotechnol 7, 2933-2937 (2007)-   Yang, H et al. Biotechnol. Bioeng. 101, 357-68 (2008)-   Yang, L et al. Proc. Natl. Acad. Sci. USA 103, 11479-84 (2006)-   Yang, L et al. Nat. Biotechnol. 26(3), 326-34 (2008)-   Zacharias D A. Science 296, 913-916 (2002)-   Zaccharias D A. Science's STKE 131, PE23 (2002)-   Ziegler, L et al. Hum Gene Ther. 19(9), 861-72 (2008)

The invention claimed is:
 1. A method for modifying the envelopecomposition of an enveloped viral particle, comprising the steps a)concentration of isolated viral particles from a suspension fluid; b)incubation of the concentrated viral particles with one or morereactants consisting of a hydrophilic target domain and a lipophilicmembrane anchor domain, wherein the lipophilic membrane anchor domainbecomes integrated into the lipid double layer of the envelope andwherein the hydrophilic target domain becomes exposed to the suspensionfluid; and c) separation of envelope modified viral particles fromexcessive reactants.
 2. The method according to claim 1 furthercomprising d) detection of the envelope modified viral particles.
 3. Themethod according to claim 1, wherein the viral envelope has a protein tolipid ratio between 50:50 and 90:10 mol %.
 4. The method according toclaim 1, wherein the lipophilic membrane anchor domain is selected fromthe group consisting of phospholipid-polyethyleneglycol, stearyl,palmityl, myristyl, cholesterol, chelator lipid nnitrilotriacetic acidditetradecylamine (NTA-DTDA) and glycosylphosphatidylinositol (GPI). 5.The method according to claim 1, wherein the hydrophilic target domainis selected from the group consisting of polysaccharides, nucleic acids,dyes, radioactive ligands, fluorescent dyes, synthetic beads andmagnetic particles.
 6. The method according to claim 1, wherein thehydrophilic target domain is a protein or a polypeptide.
 7. The methodaccording to claim 6, wherein the protein or the polypeptide furthercomprises a protein tag.
 8. The method according to claim 6, wherein theprotein or the polypeptide is an enzyme, an antibody, a receptor, amarker protein, a fluorescence protein, a complement inhibitor or acytokine.
 9. The method according to claim 1, wherein the incubationstep is achieved by slow agitation in body fluid, cell culture medium,buffered saline or physiological saline at temperatures between 4 to 40°C. for 5 minutes to 48 hours.
 10. The method according to claim 1,wherein the enveloped viral particle is selected from the groupconsisting of Arenaviridae, Bunyaviridae, Coronaviridae, Filoviridae,Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae,Paramyxoviridae, Poxviridae, Retroviridae, Rhabdoviridae andTogaviridae.
 11. The method according to claim 1, wherein the envelopedviral particle is inactivated or attenuated.
 12. The method according toclaim 10, wherein the enveloped viral particle is one of a retrovirus, apoxvirus, a herpesvirus and a lentivirus.
 13. The method according toclaim 12, wherein the enveloped viral particle is one of a mouseleukemia virus, a feline herpesvirus and a vaccinia virus.
 14. Themethod according to claim 1, wherein viral particle further comprises agenetically modified genome compared to its wild-type form.